Probing the Role of Insulin-Like Growth Factor-Binding Protein 3 and Humanin in Regulating Hyaluronan Function

项目总结 众所周知，蛋白质在许多细胞过程中起着至关重要的作用， 人类疾病是由于它们的功能被破坏而引起的。然而，知识上的许多空白需要填补，以 更好地了解蛋白质如何调节和微调糖胺多聚糖(GAG)的信号转导， 高度动态的细胞外基质的成分。胰岛素样生长因子结合蛋白3(IGFBP-3)是 已知结合某些GAG，并在胰岛素样生长因子(IGF)依赖或非依赖的情况下工作 发挥其细胞毒作用的方式。IGFBP-3-GAGS相互作用的失调是细胞毒作用的基础 机制，在很大程度上是未知的，需要阐明，目前代表着我们的 知识。我们的长期目标是开发工具来开启和关闭蛋白质-GAG相互作用以调节 细胞存活。这项提案的总体目标是阐明IGFBP-3在 透明质酸(HA)和硫酸乙酰肝素(HS)信号的调制，以及最终如何调制该信号 会导致细胞毒性。我们的中心假设是IGFBP-3在调节多种方面的过程中起着关键和至关重要的作用 恶作剧信号。实现我们在这项提议中的目标将导致对分子的新见解 基础蛋白质-GAG相互作用的机制。我们的具体目标是：1.检验假设 IGFBP-3阻断HA-CD44信号导致其自身水平和信号依赖于P53的增加， 以及依赖于p53的乙酰肝素酶水平下降，促进细胞凋亡，降低细胞存活率， 2.验证细菌附着和对接到哺乳动物细胞表面的假设 胞外IGFBP-3与HS的结合和乙酰肝素酶的活性，3.检验假说 用HA合成抑制剂4-甲基伞形酮(4-MU)或IGFBP-1阻断HA-CD44信号转导 3，导致基质金属蛋白酶-2和-9(MMP2/9)水平下降，细胞外增加 现在能够与其受体结合的Pro-BDNF的积累，降低了细胞存活率，4.检验假设 酪蛋白激酶2(CK2)对IGFBP-3的磷酸化可通过两种机制提高细胞存活率： A)磷酸化抑制IGFBP-3‘S与HA的结合能力，允许糖重新结合其受体CD44；b) 抑制细胞保护肽人蛋白(HN)与IGFBP-3的结合，使HN与淀粉样蛋白-β结合 多肽(Aβ)，募集乙酰胆碱酯酶(AChE)形成三元复合体，降低AChE活性， 增加ACh水平，激活α7nAChR。更好地理解这些基本机制将 提高我们对蛋白质-多肽-碳水化合物信号转导失调引起的疾病的认识 很可能为针对蛋白质-GAG相互作用的新治疗策略提供有价值的线索 基本的细胞过程。此R15应用程序为介绍本科生提供了一个有效的工具 和研究生在他们的早期阶段接受真实和广泛的动手研究培训 教育和培养自信、适应力、对生物医学研究事业的欣赏和兴趣。

PROJECT SUMMARY Proteins are known to play a crucial role in the undertaking of numerous cellular processes and a variety of human diseases result from disruption of their function. Many gaps in knowledge need to be filled, however, to gain a better understanding of how proteins regulate and fine-tune signaling of glycosaminoglycans (GAGs), components of the highly dynamic extracellular matrix. Insulin-like growth factor-binding protein-3 (IGFBP-3) is known to bind certain GAGs and operate in either an insulin-like growth factor (IGF)-dependent or independent manner to exert its cytotoxic functions. How dysregulation of the IGFBP-3-GAGs interactions underlies cytotoxic mechanisms, is largely unknown, needs to be elucidated, and currently represents a significant gap in our knowledge. Our long-term goal is to develop tools to switch protein-GAGs interactions ‘on and off’ to regulate cell survival. The overall objective of this proposal is to shed light on the mechanisms employed by IGFBP-3 in modulating hyaluronan (HA) and heparan sulfate (HS) signaling, and how modulation of this signaling ultimately leads to cytotoxicity. Our central hypothesis is that IGFBP-3 is a key and vital player in regulating diverse aspects of GAG signaling. Accomplishing our aims in this proposal will lead to novel insights into the molecular mechanisms underlying fundamental protein-GAGs interactions. Our specific aims are to: 1. Test the hypothesis that IGFBP-3 blocks HA-CD44 signaling resulting in a p53-dependent increase in its own levels and signaling, and a p53-dependent decrease in the levels of heparanase, promoting apoptosis and decreasing cell survival, 2. Test the hypothesis that bacterial attachment and docking to the mammalian cell surface is hindered by both binding of extracellular IGFBP-3 to HS and by the enzymatic activity of heparanase, 3. Examine the hypothesis that blocking HA-CD44 signaling with either the HA synthesis inhibitor 4-Methylumbelliferone (4-MU) or IGFBP- 3, leads to decreased matrix metalloproteinases-2 and -9 (MMP2/9) levels and increased extracellular accumulation of Pro-BDNF which is now able to bind its receptor, decreasing cell survival, 4. Test the hypothesis that phosphorylation of IGFBP-3 by casein kinase 2 (CK2) results in increased cell survival by two mechanisms: phosphorylation a) inhibits IGFBP-3’s ability to bind HA allowing the sugar to re-bind its receptor, CD44; b) inhibits binding of the cytoprotective peptide, humanin (HN) to IGFBP-3, allowing HN to bind the amyloid-β peptide (Aβ), recruiting acetylcholinesterase (AChE) into a ternary complex, decreasing AChE activity, increasing ACh levels, and activating the α7nAChR. Better understanding of these basic mechanisms will advance our knowledge of diseases resulting from dysregulation of protein-peptide-carbohydrate signaling and is likely to provide valuable clues into novel therapeutic strategies targeting protein-GAGs interactions in fundamental cellular processes. This R15 application provides an effective vehicle for introducing undergraduate and graduate students to an authentic and extensive hands-on research training at an early stage of their education and cultivates confidence, resilience, appreciation for and interest in biomedical research careers.